1. Volume 124, Issue 5, Pages 1465-1475 (May 2003)
Expression of hepatitis c virus proteins inhibits interferon α signaling in the liver of transgenic mice 
Alex Blindenbacher, Francois H.T. Duong, Lukas Hunziker, Simone T.D. Stutvoet, Xueya Wang, Luigi Terracciano, Darius Moradpour, Hubert E. Blum, Tonino Alonzi, Marco Tripodi, Nicola La Monica, Markus H. Heim 
Gastroenterology 
Volume 124, Issue 5, Pages (May 2003)
DOI: /S (03)

2. Figure 1
Expression of HCV core protein in liver cells of B6HCV mice. (A ) Formalin-fixed liver sections of C57BL/6 and of B6HCV mice were stained with hematoxylin-eosin according to standard procedures. Neither inflammatory infiltrates nor liver steatosis is present in these 14-week-old animals. The yellow size bars indicate 100μm. (B) Liver-specific expression of the A1AT-HCV transgene. Northern blot analysis was performed using RNA extracted from 1-month-old transgenic mouse tissues probed with a HCV-specific probe. Twenty μg of total RNA were loaded per lane. A probe for the 28S ribosomal RNA was used as an internal control. Sg, salivary glands; M, muscle; Sk, skin; H, heart; K, kidney; Sp, spleen; L, liver of transgenic mice; C, control (liver of wild-type mouse). (C ) Liver cell extract from a C57BL/6 (lane 1) and a B6HCV mouse (lane 2) were analyzed for the presence of HCV core protein by Western blot analysis. The asterisk (*) indicates the core protein. The positions of the molecular weight markers are indicated to the left. (D) HCV core protein can be detected by immunostaining in liver cells of B6HCV mice. Core protein is present both in the cytoplasm and in the nucleus (arrows). The size bars indicate 20μm.
Gastroenterology  , DOI: ( /S (03) )

3. Figure 2
Activation of STATs after IV cytokine injections in B6HCV and C57BL/6 mice. (A ) Nuclear extracts from liver cells were tested by EMSA with the interferon-stimulated response elements oligonucleotide. An ISGF3 shift (arrow) is readily detected in C57BL/6 mice injected with 1000 IU mIFNα/g body weight, but not in B6HCV mice. C57BL/6 and B6HCV mice were injected with PBS, 100 IU mIFNα/g body weight, or 1000 IU mIFNα/g body weight as indicated. (B) EMSA with the m67 oligonucleotide. The positions of gel shift complexes containing STAT3 homodimers, STAT1-STAT3 heterodimers, and STAT1 homodimers are indicated. No induction of STAT gel shift complexes can be detected in both C57BL/6 and B6HCV mice after injection with 250 IU mIFNα/g body weight (lanes 1 and 2, respectively). Injection of 1000 IU mIFNα/g body weight induces STAT dimers in a C57BL/6 mouse (lane 3) but not in a B6HCV mouse (lane 4). In both the C57BL/6 (lane 5) and the B6HCV mouse (lane 6), STAT gel shifts are induced by 4000 IU mIFNα/g body weight. (C ) TNFα-induced NFκB activation is not impaired in B6HCV mice. EMSA with NFκB consensus oligonucleotide. Injection of 5 ng TNFα/g body weight results in NFκB shifts (arrow) in both a C57BL/6 mouse (lane 1) and in two B6HCV mice (lanes 2 and 3). PBS controls are shown in lane 4 (C57BL/6) and lane 5 (B6HCV).
Gastroenterology  , DOI: ( /S (03) )

4. Figure 3
Tyrosine phosphorylation and nuclear translocation of STATs. (A ) Tyrosine phosphorylation of STATs is not impaired in B6HCV mice compared with C57BL/6 mice. Mice were injected IV with PBS or with 1000 IU mIFNα/g body weight and euthanized 20 minutes later. The arrows indicate STAT1 and STAT3, respectively. The position of the 116 kilodalton and 85 kilodalton size markers is shown. (B) Nuclear import of phospho-STAT1 is not inhibited in B6HCV mice. Cytoplasmic as well as nuclear extracts were prepared from livers of control mice and HCV transgenic mice 20 minutes after injection with PBS or 1000 IU mIFNα/g body weight as indicated. Tyrosine phosphorylated STAT1 was detected by Western blot analysis with phospho-STAT1–specific antibodies. (C ) Phospho-STAT1 nuclear translocation is impaired in B6HCV mice. The scale bar represents 50 μm. Hepatocytes with phospho-STAT1 nuclear localization were counted under a fluorescent microscope. Five C57BL/6 mice and 4 B6HCV mice were analyzed. For each animal, 10 areas were randomly chosen for counting. About 2000 cells were counted per animal. The histogram shows the average percentage of cell nuclei that stain positive with the phospho-STAT1–specific antibody 9171S in C57BL/6 and B6HCV mice, respectively. The error bars represent the 95% confidence interval for the mean values.
Gastroenterology  , DOI: ( /S (03) )

5. Figure 4
Increased susceptibility of B6HCV mice to LCMV-WE infection. (A ) B6HCV (red circles) and C57BL/6 (blue rectangles) mice were injected IV with the indicated doses of LCMV-WE. Serum ALT values (U/L) were determined at day 0, 4, 8, and 12. Three mice each were used per group and per LCMV-WE dose. The rectangles and circles represent mean values; the bars show the standard error of the mean. (B) All C57BL/6 and B6HCV mice were euthanatized 12 days after IV injection of 2 × 105 pfu of LCMV. Formalin-fixed liver sections were stained with H&E according to standard procedures. Representative samples are shown. Whereas C57BL/6 mice show no signs of hepatitis, a moderately severe hepatitis with slight lobular disarray, diffuse intrasinusoidal mononuclear infiltrates, and apoptotic hepatocytes (arrow) are seen in B6HCV mice. The yellow size bars indicate 100 μm. (C ) Virus titers were measured 12 days after infection with the indicated doses. Results from B6HCV mice are shown in red, controls in blue. Two mice each were used per group and LCMV-WE dose. The bars are standard error of the means. The assay has a limit of detection at 4.2 log 10 pfu (shown as yellow bar). After injection of 2 × 103 pfu in both groups and after injection of 2 × 104 pfu in controls, the values were below the limit of detection.
Gastroenterology  , DOI: ( /S (03) )

6. Figure 5
Double immunostaining for HCV core protein and phospho-STAT1 in C57BL/6 and B6HCV after injection of mIFNα. (A ) Hoechst stain of C57BL/6 mouse liver section. (B) Hoechst stain of B6HCV mouse liver section. (C ) phospho-STAT1 staining, C57BL/6, (D) phospho-STAT1 staining, B6HCV. (E ) HCV core protein staining, C57BL/6, (F ) HCV core protein staining, B6HCV. As expected, no HCV core protein could be detected in C57BL/6 mice. In B6HCV mice, approximately 30% of hepatocytes show HCV core protein nuclear localization and its presence in the nucleus is negatively correlated with the presence of activated STAT1. The white arrows show nuclei of hepatocytes that stain positive for HCV core but negative for phospho-STAT1. The blue arrows show hepatocytes with nuclear staining for phospho-STAT1 but without nuclear staining for HCV core. The yellow size bar indicates 20μm.
Gastroenterology  , DOI: ( /S (03) )